Systematic Knockdown of Epigenetic Enzymes Identifies a Novel Histone Demethylase PHF8 Overexpressed in Prostate Cancer with An
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Oncogene (2012) 31, 3444–3456 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Systematic knockdown of epigenetic enzymes identifies a novel histone demethylase PHF8 overexpressed in prostate cancer with an impact on cell proliferation, migration and invasion M Bjo¨rkman1,PO¨stling1,2,VHa¨rma¨1, J Virtanen1, J-P Mpindi1,2, J Rantala1,4, T Mirtti2, T Vesterinen2, M Lundin2, A Sankila2, A Rannikko3, E Kaivanto1, P Kohonen1, O Kallioniemi1,2,5 and M Nees1,5 1Medical Biotechnology, VTT Technical Research Centre of Finland, and Center for Biotechnology, University of Turku and A˚bo Akademi University, Turku, Finland; 2Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland and 3HUSLAB, Department of Urology, University of Helsinki, Helsinki, Finland Our understanding of key epigenetic regulators involved in finger proteins like PHF8, are activated in subsets of specific biological processes and cancers is still incom- PrCa’s and promote cancer relevant phenotypes. plete, despite great progress in genome-wide studies of the Oncogene (2012) 31, 3444–3456; doi:10.1038/onc.2011.512; epigenome. Here, we carried out a systematic, genome- published online 28 November 2011 wide analysis of the functional significance of 615 epigenetic proteins in prostate cancer (PrCa) cells. We Keywords: prostate cancer; epigenetics; histone used the high-content cell-spot microarray technology and demethylase; migration; invasion; PHF8 siRNA silencing of PrCa cell lines for functional screening of cell proliferation, survival, androgen receptor (AR) expression, histone methylation and acetylation. Our study highlights subsets of epigenetic enzymes influencing different cancer cell phenotypes. Plant homeo domain Introduction (PHD) finger proteins have a key role in cell survival and histone methylation, whereas histone deacetylases were Cancer initiation and progression have been recognized primarily involved in regulating AR expression. In as complex processes that involve both genetic and contrast, JumonjiC-domain (JmjC) containing histone epigenetic alterations (Sharma et al., 2010). Epigenetic lysine demethylases (KDMs) mainly had an impact on cell changes in cancer progenitor cells may represent early or proliferation. Our results show that the KDMs JARID1B, initial steps in tumorigenesis, resulting in polyclonal PHF8, KDM3A, KDM3B and KDM4A were highly precursor populations prone to the accumulation of expressed in clinical PrCa samples. The PHD-finger additional genetic and epigenetic defects (Holst et al., protein 8 (PHF8), a transcriptional coactivator with both 2003). Furthermore, global changes in histone modifica- PHD- and JmjC-domains, was moderately to strongly tion patterns are functionally associated with cancer expressed in 80% of clinical PrCa samples, whereas 76% development and recurrence (Fraga et al., 2005; Seligson of normal and benign samples were negative or only et al., 2005, 2009). These epigenetic modifications are showed weak PHF8 expression. Strong PHF8 expression mediated by antagonizing sets of enzymatic complexes: correlated significantly with high Gleason grade and was the ‘writer’ proteins, which attach chromatin modifica- borderline significant for poor prognosis. The results of tions in a site-specific manner, and the ‘eraser’ proteins functional PHF8 knockdown implicate a role in cell that remove these (Ruthenburg et al., 2007). Such migration and invasion, as shown by cell motility and 3-D modifications are interpreted by ‘reader’ proteins that invasion assays. Our study suggests that various cellular specifically bind to the modified chromatin. These phenotypes are regulated by distinct subsets of epigenetic complexes direct transcriptional regulator complexes enzymes. Proteins interpreting and modifying histone to active promoter sites. Aberrant expression of methylation, such as JmjC-domain and particularly PHD- epigenetic targets, including DNA methyltransferases, methyl-CpG-binding proteins, histone lysine acetyl- transferases, histone deacetylases (HDACs), histone lysine methyltransferases, histone lysine demethylases Correspondence: Dr M Nees, Medical Biotechnology, VTT Technical (KDMs) and Polycomb group proteins mediate cancer Research Centre of Finland, and Center for Biotechnology, Institute for Molecular Medicine Finland and University of Turku and A˚bo development and progression (Ellis et al., 2009). A Akademi University, Turku, Varsinais Suomi FI-20520, Finland. spectrum of diverse DNA methyltransferase and HDAC E-mail: matthias.nees@vtt.fi inhibitors are currently in clinical development, whereas 4Present address: OHSU Knight Cancer Institute, Oregon Health & some have already been approved as anticancer drugs Science University, Portland, OR 97239, USA (Egger et al., 2004; Minucci and Pelicci, 2006), and 5These authors contributed equally to this work. Received 22 March 2011; revised 27 September 2011; accepted 2 October represent the first examples for a novel concept of 2011; published online 28 November 2011 therapeutics. PHF8 mediates cell migration in prostate cancer MBjo¨rkman et al 3445 Prostate cancer (PrCa) represents the third most Ontology (GO) annotations related to epigenetics, chro- common cause of cancer deaths among Western men, matin remodelling/maintenance, and co-regulatory func- primarily because of the development of castration- tions. We included genes that contained at least one key resistant metastasizing cancer. Substantial evidence epigenetic protein domain (for example, SET, bromo, supports the involvement of epigenetic processes in JmjC, plant homeo domain (PHD), HDAC or chromo). PrCa progression (Seligson et al, 2005). At the DNA Additional candidates were selected based on literature level, multiple tumor suppressor genes involved in meta-searches and protein–protein-interaction data of regulation of prostate differentiation, proliferation or epigenetic transcriptional regulator complexes. This con- metastasis are effectively silenced by promoter methyla- certed approach defined a set of 615 human genes with tion; frequently detected already in premalignant lesions known or assumed epigenetic activity (Figure 1a, Supple- (Cooper and Foster, 2009). Furthermore, global chro- mentary Table 1). In combination with a selection of matin methylation and acetylation studies have revealed controls, each gene was targeted by two independent histone modification patterns that may be predictive for siRNAs, resulting in a library of 1328 siRNAs. This PrCa recurrence (Ellinger et al., 2010; Bianco-Miotto library was spotted on plastic cell culture carriers and et al., 2010). For example, increased global levels of screened by the cell-spot microarray technique (Rantala Histone H3 lysine 4 dimethylation and H3K18 acetyla- et al., 2010; Rantala et al, 2011) in VCaP cells. The impact tion are statistically linked to an increased risk of tumor of silencing on PrCa growth (Ki67), survival (cPARP) and recurrence (Bianco-Miotto et al., 2010). In line with this, expression of AR were addressed by high-content epigenetic enzymes interpreting and modifying histones immunofluorescence staining and imaging, using specific are often aberrantly expressed in PrCa (Cooper and antibodies (Figure 1b). Additionally, altered global levels Foster, 2009), such as class I HDACs, which are of H3K4 and H3K9 dimethylation and H3K18 and overexpressed in aggressive and hormone-refractory H4K16 acetylation, linked to poor cancer prognosis, were PrCa, thus contributing to androgen-independent, lethal studied (Fraga et al., 2005; Seligson et al., 2009) PrCa’s (Halkidou et al., 2004; Weichert et al., 2008). (Figure 1b). The functional effects of siRNAs was Eight KDMs are highly expressed in PrCa’s (Metzger considered significant if the difference of staining intensity et al., 2005; Kahl et al., 2006; Wissmann et al., 2007). exceeded a Z-score 4 þ 2 s.d., compared with the median Various HDACs and KDMs were shown to regulate of scrambled control siRNAs. This resulted in the androgen receptor (AR)-mediated transcription, thus identification of 272 siRNAs, targeting 231 genes affecting affecting gene expression patterns essential for PrCa the various endpoints (Supplementary Figure S1). GO proliferation, development and progression (Yamane enrichment analysis of primary hits was used to select et al., 2006; Wissmann et al., 2007; Wolf et al., 2007; significantly over-represented protein families for second- Xiang et al., 2007; Welsbie et al., 2009). Despite the ary screening (DAVID; http://david.abcc.ncifcrf.gov/). emerging role of epigenetic processes and large scale mapping of cancer-associated chromatin modifications in Clustering of cell-spot microarray screening data by PrCa, the functional significance of key ‘readers’, ‘writers’ portioning around medoids (PAM) and ‘erasers’ has not been systematically evaluated. In the Our large-scale primary screen was primarily designed present study, we have systematically screened the func- to identify epigenetic gene classes that significantly affect tional role of 615 epigenetic genes representing all known PrCa cell survival, proliferation, AR expression and and putative epigenetically active players in the human histone modifications, but at this stage not focused on genome by siRNA knockdown. Our multiplexed cell-based identification of single candidate genes. Therefore, the screens addressed proliferation, apoptosis, expression of Z-scores of hit siRNAs from all the seven immune AR, and global histone